Identification of miRNA-rich vesicles in bronchoalveolar lavage fluid: Insights into the function and heterogeneity of extracellular vesicles

Despite emerging interest in the role of extracellular vesicle (EV)-containing microRNAs (EV-miRNAs), the existence of functional EV-miRNAs under patho-physiological conditions has been viewed with skepticism. Due to the heterogenicity of EVs, several barriers related to EV-miRNA research are to be explored before the in vivo function of EV-miRNAs can be thoroughly delineated. For example, it has been reported that far less than one copy of a given miRNA can be detected per exosome. In this study, we demonstrated that miRNA-rich-EVs exist and can be consistently isolated using differential centrifugation & density-gradient fractionation from bronchoalveolar lavage fluid (BALF) in vivo. The absolute number of this 'miRNA-rich'-EV population is only about 7.05 × 10⁹ per mouse (6% of total EVs). However, the RNA amount detected in this population of EVs represents approximately 39% of the total EV RNAs in the BALF. In contrast, the remaining populations of BALF EVs (76% of total EVs) contain extremely low concentrations of RNAs and miRNAs. The miRNA-rich-EVs in BALF are likely derived from alveolar epithelial type-I cells (ATIs). Notably, caveolin-1, a lipid raft protein, is exclusively detected in the miRNA-rich-EVs, suggesting the lipid raft protein as a biomarker of EV-miRNA enrichment. We further demonstrated that miRNAs contained in the ATI-EVs are actively delivered into alveolar macrophages, subsequently promoting inflammasome activation, neutrophil recruitment, and M1-macrophage polarization in response to P. aeruginosa pneumonia in vitro and in vivo. Collectively, we are the first to identify and characterize the miRNA-rich-EVs in BALF. These miRNA-rich EVs endorse pro-inflammatory responses in bacterial lung infection. Our study provides a novel insight into the development of biomarkers, therapeutic strategies and underlying mechanisms for lung pathology.